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Chronic toxicity and carcinogenicity studies with the insecticide endosulfan in rats and mice
~
Pergamon
0278-6915(95)00063-1
Fd Chem. Toxic. Vol. 33, No. 11, pp. 941-950, 1995 Copyright © 1995 ElsevierScienceLtd Printed in Great Britain. All fights reserved 0278-6915/95 $9.50 + 0.00
Chronic Toxicity and Carcinogenicity Studies with the Insecticide Endosulfan in Rats and Mice R. H A C K * t , E. E B E R T a n d K . - H . L E I S T Hoechst Schering AgrEvo GmbH, Toxicology and *Hoechst Aktiengesellschaft, Pharma Development Ccrporate Toxicology, D-65795 Hattersheim, Mainzer Landstrasse 500, Germany
(Accepted 16 May 1995) Abstract--The insecticide endosulfan was evaluated for chronic toxicity and carcinogenicity in long-term feeding studies in both Sprague-Dawley rats and NMRI mice. Dietary concentrations of the test substance were adminis~:ered to rats at 0, 3, 7.5, 15 and 75 ppm and to mice at 0, 2, 6 and 18 ppm for 24 months each. In the rat study, only the treatment with the highest dose caused a significant reduction of body weight gains of the males and females at 75 ppm. The increased incidence of enlarged kidneys seen at autopsy at 75 ppm in females, and the slightly increased incidence of progressive glomerulonephrosis and slightly increased incidence of renal aneurysms seen histopathologically in the 75 ppm males, make the kidney the target organ in rats. On the basis of these findings a dietary concentration of 15 ppm is considered to be the no-observed-effect level (NOEL) in rats, equivalent to a daily test substance intake of 0.6 mg/kg body weight in males and 0.7 mg/kg body weight in females. In the mouse study, the treatment with 18 ppm caused a significant increase of mortality in the females and a slight (in the first third of the study, significant) reduction of body weight gain in males. Since there were no other substance-related findings, the dietary concentration of 6 ppm is considered to be the NOEL in mice, equivalent to a daily test substance intake of 0.84 mg/kg body weight in males and 0.97 mg/kg body weight in females. An evaluation of all relevant tumour data gained in both studies revealed no differences between control and treated groups. It was concluded, therefore, that endosulfan has no carcinogenic potential.
1NTRODUCTION
strains, in a yeast forward mutation test using Schizosaccharomyces pombe, in a yeast gene convers i o n / D N A repair test using Saccharomyces cerevisiae strain D4, in a mouse lymphoma test using mouse L51786 T K ± cells, in a D N A repair test using Bacillus subtilis strain H17 and M45 (rec-), in unscheduled D N A synthesis tests using primary rat hepatocytes and the human cell line A549 and in a chromosome aberration test using cultured human lymphocytes ( W H O / F A O , 1990). There was also no indication of a mutagenic potential of endosulfan in in vivo mutagenicity tests--micronucleus tests using bone marrow of N M R I ( W H O / F A O , 1990) and Swiss Albino mice (Usha Rani et al., 1980)--and an in vivo cytogenicity test using bone marrow and spermatogonia of male albino rats (Dikshith and Datta, 1978). Endosulfan has been already investigated for possible carcinogenicity in mice and rats as part of the Carcinogenesis Testing Program of the National Cancer Institute (Innes et al., 1969; National Cancer Institute, 1978). The preliminary mouse study resuited in no significant increase in tumours (Innes et al., 1969); in the subsequent studies in mice and rats, mortality in both male mice and rats was ultimately extremely high (National Cancer Institute, 1978). For this reason, these studies were classified by
Endosulfan is an insecticide used worldwide in agriculture, viticulture and horticulture. It was first registered by Hoechst in 1958 under the trade mark Thiodan in Germany. The first registration in the United States was in 1960. After oral application to rats, endosulfan has an LDs0 from 18 to 355mg/kg body weight (WHO, 1984) or from 40 to l l 0 m g / k g body weight (IVA, 1990), depending on the sex of the animal and the vehicle used. Signs of acute intoxication include neurological manifestations, such as sedation, hyperactivity, muscular twitching, ataxia and convulsions (IVA, 1990; W H O , 1984). Endosulfan is irritating to the eyes and skin, and not sensitizing in the guinea pig (IVA, 1990). Endosulfan is not teratogenic to rats and rabbits (Gupta et al., 1978; W H O / F A O , 1990) and has no effect on rat reproduction ( W H O / F A O , 1990). The compound was not mutagenic in vitro in an Ames test using various Salmonella typhimurium tAuthor for correspondence. Abbreviations: GJIC = gap junctional intercellular communication; GOT = glutamic--oxaloacetic transaminase; GPT= glutamic-pyruvic transaminase; NOEL = noobserved-effect level. 941
942
R. Hack et al.
the NTP (Yang et al., 1989) and W H O / F A O (1990) as inadequate for assessing the carcinogenic potential of endosulfan. The purpose of this paper is to present the data of valid up-to-date studies that were performed to find a sound basis for evaluating the chronic toxicity and the carcinogenic potential of endosulfan. MATERIALS AND METHODS
Test substance
Endosulfan [6,7,8,9,10,10-hexachloro-l,4,5a,6,9,9ahexahydro - 6,9- methano- 2,4,3 - benzo- dioxathiepin3-oxide (IUPAC; CAS Reg. No. 115-29-7)] was supplied by the Agricultural Division, Hoechst Aktiengesellschaft and identified by analysis certificates for the content of active ingredient and impurities (see Table 1). Both studies were performed with the same batch. The stability was confirmed by regular re-analysis. None of the analyses revealed a content of less than 96.7% (w/w). Rat study
The study was performed at Huntingdon Research Centre Ltd (Huntingdon, UK). Sprague-Dawley rats [stock: Crl:Cd (SD) BR], were obtained from Charles River (Portage, MI, USA). They were approximately 6 wk old at the start of treatment and had a mean body weight, after a 1-wk adaptation period, of 129 g (males) and 109 g (females). The animals were housed five to a cage on a wire mesh floor. The doses used throughout this study were based on the results of a 13-wk feeding study (Barnard et al., 1985; WHO/FAO, 1990) and on a multigeneration study (Edwards et al., 1984; WHO/FAO, 1990).
In the 13-wk feeding study, Sprague-Dawley rats [stock: CrI:CD (SD) BR] received a diet containing 0, 10, 30, 60 and 360ppm endosulfan, equivalent to daily doses of 0.64, 1.92, 3.85 and 23.41 mg/kg body weight in males and 0.75, 2.26, 4.59 and 27.17 mg/kg body weight in females. From 60ppm onwards, slightly reduced body weight gains, haematological changes and increases of relative kidney weights (males, females at 360 ppm) were observed. In the multigeneration study, feeding of Sprague-Dawley rats [stock: Crl:CD (SD) BR] with daily concentrations of 0, 3, 15 and 75 ppm endosulfan, equivalent to approximately 0.2, 1.0 and 6.0 mg/kg body weight, resulted in no clinical findings in any group and in slight but not statistically significant impairment of body weight gains in the 75 ppm group. The maximum tolerated dose, based on the reduction of body weight gain (US EPA, 1988), was considered to be around 75 ppm when administered for 104 wk. The study design consisted of five groups (including one untreated control group) that were fed endosulfan technical grade with the daily diet (Labsure Animal Diet No. 2, SDS Diets Ltd, Witham, UK) in concentrations of 0, 3, 7.5, 15 and 75 ppm for 104 wk. One premix was prepared each week by dissolving the test material in acetone (final concentration in the premix, 0.4%), mixing the acetone with corn oil (final concentration in the premix, 2%) and mixing the acetone-corn oil with basal diet. The acetone was removed from the premix using a rotary evaporator at a temperature of 40°C, the mixture being left in the rotary evaporator flask until a constant weight was achieved. All the treated diet was made from the premix containing the test material, corn oil and acetone (the acetone having theoretically been
Table 1. Characterizationof the test substance used throughout both studies Common name: Chemical name (IUPAC) CAS Reg. No. Content [% (w/w)]* Analysis of the active ingredient:
Endosulfan: 6,7,8,9,10-Hexachloro-1,4,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepin-3-oxide
115-29-7
96.7-97.9
Endosulfan-ether: 4,5,6,7,8,8-Hexachloro-1,3,3a,4,7,7a-hexahydro-4,7-methano-isobenzofurane
3369-52-6
0.2-0.3
Endosulfan-diol: 1,4,5,6,7,7-Hexachloro-bicyclo[2.2.1.]bept-5-ene-2,3-dimenthanol
2157-19-9
0.2-0.3
Analysis of impurities:
Endosulfan-lactone: 3868-61-9 4,5,6,7,8,8-Hexachloro-1,3,3a,4,7,7a-hexahydro-4,7-methano-isobenzofurane-l-one Endosulfan-acetate: 1,4,5,6,7,7-Hexachloro-bicyclo[2.2.1.]bept-5-ene-2,3-dimethyl-diacetate
18992-36-4
0.4 < 0.2
Octachlorocyclopentene
--
0.1- < 0.1
Solvent residues(e.g. toluene)
--
0.2-<0.1
NaCI (measuredas chloride)
--
<0.05
Epoxidizedsoybeanoil
--
0.33-0.34
*Range of several analyses.
Endosulfan: chronic toxicity and carcinogenicity i
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During the first 4 wk, all animals were observed daily for clinical signs of toxicity, and subsequently once weekly. Examination for palpable masses was carried out every week. Mortality was recorded twice daily. Body weights were determined pre-test, at the beginning of treatment and once a week thereafter. Food consumption was measured weekly and water consumption during wk 11, 26 and 50 (chronic toxicity groups only). Ophthalmoscopic examination was carried out pre-test and during wk 50 and 104 in all animals of the control and high-concentration groups. Clinical laboratory investigations were performed in the chronic toxicity group only; for the last examinations, the sample size of 10 animals per sex was reached, where necessary, by replenishment with animals from the tumorigenicity groups. Blood samples were collected (chronic toxicity groups only) from the orbital sinus during wk 13, 26, 52, 78 and 103 after food removal overnight, and analysed for haematocrit, haemoglobin, red blood cell count, platelet count, total and differential white blood cell count, thrombin time, total protein, albumin, globulin, urea nitrogen, creatinine, sodium, potassium, calcium, inorganic phosphorus, chloride, glucose, alkaline phosphatase, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), cholesterol and total bilirubin. Urine was collected during wk 12, 25, 51, 77 and 102 after water removal
Fig. 1. Percentage survival of (a) male and (b) female rats fed endosulfan [0 ( - - ) , 3(. . . . ), 7.5 (. . . . . ), 15 (-.-) or 75 ppm (. . . . )] in the diet for 24 months.
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completely removed) by direct dilution of the premix with further amounts of standard diet for a minimum of 7 min in a double cone blender. The amount of premix used increased from the lowest-concentration diet to the high-concentration diet. The amount of corn oil (and the hypothetical amount of acetone remaining if none had been removed) in the highest concentration diet was calculated. For the control diet, a premix containing basal diet corn oil at 2% and acetone at 0.4% was prepared such that the concentration of corn oil and the hypothetical concentration of acetone in the final control and high-concentration diets never exceeded a value of 0.5% for corn oil ',and 0.1% for acetone. The final diets were stored at room temperature in the animal rooms. The stability, homogeneity and accuracy of mixing were determined prior to the start of treatment and checked in the first week and after 3monthly intervals. A constant concentration was administered to the treated groups throughout the study. Each group consisted of 50 animals per sex, intended primarily fc,r tumorigenicity evaluation. For the purpose of evaluating chronic toxicity, a further 20 rats per sex per dose were scheduled for blood sampling at scheduled intervals and for killing after 104 wk of treatment and were therefore not included in the tumorigenicity evaluation.
943
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Fig. 2. Percentage survival of (a) male and (b) female mice fed endosulfan [0 ( - - - ) , 2(. . . . ), 6 (. . . . ) or 18 ppm (-. -)] in the diet for 104wk.
944
R. Hack et al. Table 2. Statistical analysis of body weight changes of rats fed endosulfan for a period of 24 months Body weight changes (g) Male dose groups (ppm)
Treatment period (wk)
0
3
7.5
Female dose groups (ppm) 15
75
0-6
230 (30.0) --
233 (33.3) [101]
234 (36.7) l 102]
226 (30.6) [98]
15
229 (30.7) [I 00]
75
113 (20.7) --
0
110 (18.3) [96]
3
113 (17.6) [100]
7.5
108 (16.6) [96]
104"* (15.5) [92]
6-18
160 (28.0) --
157 (31.8) [98]
159 (38.7) [99]
145"* (32.2) [91]
144"* (31.6) [901
69 (17.2) --
66 (19.0) [96]
69 (19.2) [100l
68 (18.2) 199]
65 (15.0) I94]
18~4
200 (55.9) --
203 (72.8) [102]
194 (76.9) [97]
193 (74.5) [97]
163"* (76.5) [82]
153 (67.4) --
146 (57.7) [95]
153 (59.6) [100]
143 (57.3) [93]
125"* (61.7) [82]
0--64
590 (91.5) --
594 (109.3) [101]
586 (127.6) [99]
564 (107.8) [96]
536** (105.8) [91]
337 (88.1) --
322 (79.3) 196]
334 (82.6) [99]
318 (77.4) [94]
294** (76.3) [87]
64-104
- 11 (103.4)
13 (99.7)
-9 (113.0)
9 (102.4)
-43 (107.3)
94 (117.1)
69 (101.9)
80 (67.4)
82 (76.9)
56 (64.6)
0-104
580 (124.0) --
570 (125.4) [98]
531 (131.3) [92]
525 (115.4) [911
479** (91.4) 183]
398 (104.7) --
350 (107.3) [88]
414 (84.7) [104]
363 (91.9) [91]
328* (100.0) [82]
Values are means (g); +standard deviation in parentheses; percentage of control in square brackets. Asterisks indicate significant differences from control (*P < 0.05; **P < 0.01; Williams' test).
overnight, and analysed for volume, pH, specific gravity, protein, total reducing substances, glucose, ketones, bile pigments, urobilinogen, haem pigments and sediment. Complete gross autopsies were performed on all animals. Selected organs were weighed. Tissues-including macroscopically abnormal tissues from all animals of the control and high-dose group, tissues of all animals that had died or were killed in e x t r e m i s during the course of the study, any macroscopically abnormal tissue in any group, and lungs, liver and kidneys of all groups at terminal killing--were fixed in 10% formaldehyde solution and embedded in paraffin wax. Section cut at 4/zm were stained with haematoxylin and eosin; in addition, frozen sections of liver fixed in 10% formaldehyde solution and cut at 12 #m were stained for fat with Oil Red O.
4 wk and a mean body weight after a 1-wk adaptation period of 23 g (males) and 22.5g (females). The animals were kept individually in Macrolon cages (type I) with wire mesh tops, with standardized and sterilized granulated softwood bedding. The doses used throughout this study were based on the results of a 3-month subchronic oral toxicity study (Barnard et al., 1984; WHO/FAO, 1990) and of a supplementary 6-wk feeding study (Donaubauer et al., 1985; WHO/FAO, 1990). In the subchronic study CD-I mice [stock: CrI:CD-I(ICR)BR] were fed diets containing the test material at doses of 2, 6, 18 or 54ppm, equivalent to 0.24, 0.74, 2.13 and 7.3 mg/kg body weight in males and 0.27, 0.80, 2.39 and 7.5 mg/kg body weight in females. Mice receiving 54 ppm exhibited convulsions and salivation, and 12 of 20 males and 10 of 20 females died within 6 wk of study initiation. No mortality or other toxic effects were observed at lower levels. The 6-wk study with NMRI mice [stock: Hoe:NMRKf (SPF71)] given 18 ppm with the diet, equivalent to 3.7 and 4.6 mg/kg body weight in males and females, respectively, was performed to check the sensitivity of the NMRI mice to endosulfan. The duration was limited to 6 wk since
M o u s e study
The study was performed at Hoechst Aktiengesellschaft, Pharma Development Corporate Toxicology, Frankfurt, Germany. NMRI mice [stock: Hoe:NMRKf (SPF71)] from the Hoechst breeding colony were used. They had an age of about
Table 3. Relative incidence of selected non-neoplastic findings in male rats after feeding of endosulfan for 104 wk compared with historical control data
Dose groups~: (ppm) Findings
0
Progressive 0.286 glomerulonephrosis Aneurysm 0.143
3
7.5
0.257
0.314
0.342 0.428t
15
75
Historical rang~ 0.100-0.380
0.086
0.243
0.143 0.271"
0.040-0.180
*n = 70; tumorigenic + chronic toxicity group. §Range of six studies with n = 50 animals each. Asterisk and dagger indicate significant differences from controls (*P <0.05; tP < 0.06; Fisher's exact test).
E n d o s u l f a n : c h r o n i c toxicity a n d c a r c i n o g e n i c i t y
945
Table 4. Mcidence of neoplastic lesions in rats after administration of endosulfan for 104 wk (n = 50) Male dose groups (ppm)
Female dose groups (ppm)
Organ/tumour type*
0
3
7.5
15
75
0
3
7.5
15
75
Multicentric tumours Lymphosarcoraa Myeloid leukaemia Histiocytic sarcoma
0 0 0
I 0 0
0 1 0
0 0 0
0 0 0
0 0 1
0 0 0
0 0 1
2 0 0
1 0 0
Liver Haemangioma Haemangiosarcoma Malignant liver cell tumour Cholangioma
0 0 1 1
0 0 1 0
1 0 1 0
0 0 1 0
0 0 2 0
0 0 0 0
1 0 0 0
1 1 0 0
0 1 0 0
0 0 0 0
Kidney Renal liposarcoma Renal carcinoma Renal lipoma Mesenchymal tumour
1 0 0 0
0 1 0 0
1 0 0 0
0 0 1 0
l 1 0 0
1 0 0 0
0 0 0 0
0 1 0 0
0 0 0 1
0 0 0 0
Lungs Pulmonary adenocarcinoma Pulmonary adenoma
0 0
1 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 1
Brain Granulocytic meningioma Astrocytoma
0 0
1 1
0 0
0 l
0 0
0 1
0 0
0 0
0 0
0 I
Spleen Haemangioma Haemangiosarcoma Fibrosarcoma
0 0 0
0 l 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 1
0 0 0
0 0 0
1 0 0
Pancreas Islet cell carcinoma Islet cell adenoma Islet cell aderoma (two)
3 8 0
8 6 0
3 9 0
5 11 3
5 4 0
0 0 0
0 5 0
0 0 0
0 5 0
0 5 0
Thyroids Parafonicular cell carcinoma Follicular carcinoma Follicular adenoma
3 0 0
0 3 0
0 0 4
0 0 0
3 0 5
0 0 0
3 0 0
0 0 0
0 0 0
0 0 0
Adrenals Phaeochromccytoma
3
0
0
3
0
0
0
0
0
0
0 23
0 18
0 16
0 21
0 27
3 31
0 31
0 39
0 34
0 32
Skin Fibroma Dermal fibro:na Keratoacantl" oma
0 5 0
0 3 4
0 0 5
3 0 4
0 0 3
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
Prostate Prostatic adeaocarcinoma
1
1
0
0
0
Testes Interstitial cell tumour
0
2
2
2
1
Epididymides Mesothelioma
0
0
0
1
0
Uterus Fibrosarcoma Endometrial sarcoma Squamous cell carcinoma Uterine adenocarcinoma
-----
-----
1 1 0 0
1 1 0 0
0 1 0 0
0 0 0 0
0 0 I 1
Ovaries Tubular adenoma
--
--
0
2
0
1
0
Cervix Stromal sarcoma
--
--
0
0
0
0
0
Subcutaneous mass Lipoma Fibroma Mammary fibroma Mammary fibroadenoma Mammary fibroadenoma with atypia Mammary adenoma Mammary adenocarcinoma
5 3 0 2 0 0 0
7 0 0 2 0 0 0
10 3 0 1 0 0 1
3 3 0 1 0 0 1
3 3 0 0 0 0 0
0 3 0 34 8 0 14
0 4 4 34 3 3 12
0 3 0 36 4 0 14
0 4 0 29 0 3 9
0 0 5 31 3 0 10
Mammary gland Basal cell carcinoma
0
1
0
0
0
0
0
0
0
0
Pituitary Carcinoma in pars anterior Adenoma in pars anterior
m
m
m
_
E
m
_
E
m
m
*Only tumours of the main organs, possible target organs and those for which the incidence was greater than one in any of the groups are presented. Statistical significance was tested using Mante!-Haenszel's test and Fisher's exact test.
946
R. Hack et al. Table 5. Cumulative mortality and percentage survivalt in mice (n = 60) fed endosulfan for 24 months Male dose groups (ppm) Female dose groups (ppm) Study wk 0 2 6 18 0 2 6 18 1-26 3 (95) I (98) I (98) 2 (97) 3 (95) 2 (97) 0 (100) 1 (98) 27-52 5 (92) 8 (87) 10 (83) 15 (75) 4 (93) 2 (97) 8 (87) 12 (80) 53-78 1 2 ( 8 0 ) 1 4 ( 7 7 ) 1 6 ( 7 3 ) 17(72) 11 (82) 1 0 ( 8 3 ) 1 6 ( 7 3 ) 23(62) 79-end 27 (55) 33 (45) 37 (38) 35 (42) 33 (45) 36 (40) 38 (37) 43 (28)* tPercentage survival in parentheses. Asterisk indicates significantdifferencefrom control (*P < 0.05; Kaplan Meier estimate and log rank test).
the only s y m p t o m s (i.e. clinical signs of toxicity and mortality) had appeared during this period of treatm e n t in the 3 - m o n t h study. Two of 10 females fed diet containing 18 p p m died on days 28 and 38; no other effects were reported. The m a x i m u m tolerated dose based on the mortality (US EPA, 1988) was considered to be a r o u n d 18 p p m when fed for a period of 24 months. The study design consisted o f four groups (including one untreated control group) t h a t were fed endosulfan technical grade with the daily diet (maintenance diet 1321, A l t r o m i n International, Lage, G e r m a n y ) in c o n c e n t r a t i o n s of 0, 2, 6 and 18 p p m for 24 months. A premix for each dose group was prepared at 14-day intervals by dissolving the test substance in sesame oil (final concentration in the premix, 2%), then by mixing with the basal diet so that the premix c o n t a i n e d the 14-fold c o n c e n t r a t i o n of test substance of the final concentration. The final dietary mixtures were prepared fresh once weekly by direct dilution of the premixes with 13-fold a m o u n t s of the basal diet for 30 min in a p l o u g h s h a r e mixer. C o n t r o l animals received n o r m a l diet prepared from a premix with 2 % sesame oil without addition o f test substance by admixture of 13-fold the same a m o u n t o f basal diet. The final diets c o m p o s e d of test substance (not in the control), of a fixed portion of 0.14 corn oil a n d basal diet were stored at r o o m t e m p e r a t u r e in the animal rooms. The stability, concentrations a n d h o m o g e n e i t y o f mixtures were determined prior to the start of t r e a t m e n t and checked four times during the course of the study. The four groups each consisted o f 60 animals per sex a n d dose and were scheduled for terminal killing after 24 months. A further 10 animals per sex a n d
dose were scheduled for interim killing after 12 a n d 18 months, respectively for each group, and were therefore not included in the tumorigenicity evaluation. Behaviour, general health condition a n d mortality were checked twice daily (at weekends a n d on public holidays once daily). Once a week the animals were examined for neurological disturbances, opacity of the refracting media o f the eyes, i m p a i r m e n t of dental growth a n d changes in the oral mucosa. Body weights and food c o n s u m p t i o n were determined once weekly. E x a m i n a t i o n for palpable masses was performed monthly, a n d subsequently twice m o n t h l y from m o n t h 6 onwards. Blood samples were taken from 10 non-starved animals per sex a n d dose after 6 m o n t h s (intermediate, using the 12-month interim killing group), 12 m o n t h s (at interim killing), 18 m o n t h s (at interim killing) and 24 m o n t h s (at terminal killing). The samples were analysed for red blood cell count, haemoglobin, haematocrit, total a n d differential white blood cell count, platelet c o u n t a n d reticulocyte count. The serum enzymes alkaline phosphatase, G O T and G P T were m e a s u r e d after killing only. Complete gross autopsies were performed on all animals. Selected organs were weighed. T i s s u e s - including macroscopically a b n o r m a l tissues from all animals of any group, tissues of all animals that had died or were killed in extremis during the course of the study, any macroscopically a b n o r m a l tissue in any group, and lungs, liver and kidneys of all groups at terminal killing--were fixed in 10% formaldehyde solution a n d e m b e d d e d in paraffin wax. Sections cut at 4/~m were stained with haematoxylin a n d eosin.
Table 6. Representative results of mean body weightt of mice (n = 60) fed endosulfan for 24 months Male dose groups (ppm) Female dose groups (ppm) Study wk 0 2 6 18 0 2 6 18 1 27(_+2) 26(_+2) 27(_+2) 25(+_2) 23(-+1) 23(-+1) 23(_+1) 23(-+1) 13 35(_+3) 35(-+3) 35(-+3) 34(_+3)* 28(-+2) 28(_+2) 29(_+2) 29(_+2) 26 37(_+3) 37(_+3) 37(_+4) 35(_+4)* 30(_+2) 30(_+2) 30(_+2) 31 (_+2) 52 38(_+4) 38(_+4) 39(_+4) 37(-+3) 32(_+2) 32(_+2) 32(_+3) 32(_+4) 78 39(_+3) 38(_+3) 38(_+3) 37(_+4) 34(_+4) 33(_+3) 34(_+3) 34(-+2) 92 38(-+3) 38(_+3) 37(_+3) 36(_+4) 34(+3) 33(_+3) 34(_+3) 34(_+4) 104 37(_+3) 37(_+3) 36(_+3) 35(_+4) 34(_+3) 33(_+3) 34(_+4) 34(_+4) t -+ Standard deviation in parentheses. Asterisks indicate significantdifferencesfrom controls (*P < 0.05; statistical analysis was carried out with a program package including procedures such as parametric method by Dunnett or Sidak and distribution-frce method by Nemenyi/Dunnett or Nemenyi/Sidak).
947
Endosulfan: chronic toxicity and carcinogonieity Table 7. Incidence of selected neoplastic lesions in mice after administration of endosulfan for 104 wk (n = 60) Male dose groups (ppm)
Female dose groups (ppm)
0
2
6
18
0
2
6
18
11 I 3 1 0
13 1 2 0 0
18 1 1 0 0
16
22
25
25
15
1
2
3
1
1
0 0 0
2 0 0
1 0 0
0 0 I
2 0 0
0 0 0 1 1
0 0 1 0 0
1 0 0 0 0
I 2 0 1 0
0 0 0 0 0
0 0 0 0 0
0 0 1 0 0
0 0 0 1 0
Lungs Pulmonary adenoma Two pulmonary adenomas Pulmonary adenocarcinoma
10 1 2
5 0 2
5 0 2
5 0 3
2 0
3 1
2 0
2 0
1
4
2
0
Adrenals Corlical adenoma
13
12
6
10
0
0
0
0
Pituatary Adeaoma in pars anterior
0
0
0
0
6
7
1
0
Testes Inte::stitial cell tumour
2
4
1
0
Organ/l:umour type* Multice:ntdc tumours Lymphosarcoma Pleomorphic lymphosarcoma Histiacytic sarcoma Lymphoid leukaemia Myeloid leukaemia Liver Benign liver cell tumour Haemangioma Haemangiomas Haemangiosarcoma Anaplastic sarcoma
Ovaries Tubular adenoma Papillary cystadenoma Granulosa cell tumour Thecoma Lut(;al cell tumour Haemangioma Haemangiosarcoma Sex cord stromal tumour
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
5 0 8 3 9 I 0 I
3 3 I0 2 5 2 I I
2 3 II 2 2 0 0 0
4 2 13 I I 0 0 0
Uterus An~.plastic carcinoma Leiomyosarcoma End ometrial sarcoma Fibrosarcoma naemangioma Haemangiosarcoma
. . . . . .
. . . . . .
. . . . . .
. . . . . .
1
0
0
0
1
0
0
1
1
0
0
0
0 0 0
0 I 1
0 0 0
I 1 0
Subcutaneous mass Mammary adenocareinoma Mammary fibroadenoma with atypia Mammary adenoacanthoma Fibrosarcoma Haemangiosarcoma
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 I 0
1
0
3
0
0
0
1
0
1
0
0
0
0 0
0 0
0 0
0 I
*Only tumours of the main organs, possible target organs and those for which the incidence was greater that one in any of the groups are presented. Statistical significance was tested using MantelHaenszel's test and Fisher's exact test.
RESULTS
Rat study T h e m o r t a l i t y rate w a s unaffected by the t r e a t m e n t (Fig.
l). N o
treatment-related
effects o n clinical
condition, food consumption, water consumption, h a e m a t o l o g y , clinical c h e m i s t r y , urinalysis a n d o p h t h a l m o s c o p y were a p p a r e n t . T h e overall b o d y w e i g h t gain f o r rats o f either sex receiving 7 5 p p m ,
however, was lower than
that
o f c o n t r o l s d u r i n g the m a i n g r o w t h p h a s e o f the s t u d y ( w k 0--64), :an effect t h a t w a s m o r e s t r o n g l y m a r k e d in the females t h a n in the males. T h e effect FCT33/II--D
o n the g r o w t h o f m a l e s at 75 p p m w a s n o t a p p a r e n t until after w k 6, w h e r e a s the effect o n females at 75 p p m w a s n o t e d f r o m the s t a r t o f t r e a t m e n t . T h e o t h e r d o s e g r o u p s were c o n s i d e r e d n o t to be affected ( T a b l e 2). T h e r e were n o statistically significant effects o n o r g a n w e i g h t s in a n y d o s e g r o u p . T h e m a c r o s c o p i c p a t h o l o g y revealed t r e a t m e n t - r e l a t e d a l t e r a t i o n s in the 75 p p m g r o u p in the f o r m o f a g r e a t e r n u m b e r o f e n l a r g e d k i d n e y s in female rats, a g r e a t e r n u m b e r o f a n e u r y s m s in m a l e r a t s m a i n l y f r o m the c h r o n i c toxicity g r o u p , a n d a g r e a t e r n u m b e r o f e n l a r g e d l u m b a r l y m p h n o d e s w i t h n o clear-cut
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R. Hack et al.
treatment-related histopathological changes in males mainly from the chronic toxicity group. The histopathological examination revealed, in the highest male group, a minimally increased incidence of progressive glomerulonephrosis and an increased incidence of aneurysms in the kidneys compared with the control and the historical range (Table 3). The findings of macroscopic and histopathological examination make the kidney the target organ, especially in the male rat. There was no indication of any treatment-related effect on the reproductive organs. The neoplastic changes are summarized in Table 4. Among the most commonly occurring tumours were pituitary tumours in male and female rats and mammary tumours in females. The type and frequency of tumours were similar in dosed and control rats and gave no indication of a treatment-related effect. The results of this study gave no indication of any oncogenic potential of endosulfan in rats. The NOEL is 15 ppm, equivalent to a mean daily test substance intake of 0.6 mg/kg body weight in the males and 0.7 mg/kg body weight in the females. Mouse study
There were no treatment-related effects on clinical signs, food and water consumption, clinical chemistry and haematology throughout the study. No differences in the number of palpable masses between dosed and control animals were noted. The mortality rate among the females in the 18 ppm dose group, however, was significantly increased. The cumulative mortality and percentage survival data are shown in Fig. 2 and summarized in Table 5. At study termination after 106 wk the survival rate among the high-dose females was lower than that among the controls (28% v. 45%). Survival among other dosed females and all dosed males was comparable to controls, ranging from 37 to 55%. Survival among dosed animals of the satellite groups killed at 12 months was comparable with that of the controls; no deaths occurred among dosed or control animals scheduled for interim killing at 18 months (data not shown). The development of body weights is shown in Table 6. Body weights of males receiving 18 ppm were slightly but significantly decreased in the first third of the study when compared with concurrent controls; body weights of these animals remained slightly but not significantly decreased thereafter, to study termination. The body weights of other dosed males and all dosed females were similar to those of controls throughout the study. Macroscopic pathology gave no indications of intergroup differences. The organ weights after 24 months did not reveal any statistically significant differences between control and treated animals. At 12-month interim killing the relative lung and ovary weights in the top-dosed females, and after 18-month interim killing the relative liver weights of the topdosed males and ovary weights of the top-dosed
females, were lowered. All these values, however, were within the normal range of this stock of mice and were therefore considered as incidental (data not shown). The histopathological examination revealed no treatment-related changes at any killing point or for any sex and dose. The neoplastic findings are listed in Table 7. The most commonly observed neoplasms were lymphosarcomas and pulmonary adenomas in both sexes, adrenal cortical adenomas in male and granulosa cell tumours and luteal cell tumours in females. The incidence of findings was similar in dosed and control animals. There was no indication of any effect on reproductive organs. All findings were considered to be of a normal age- and stockrelated nature. The results of this study gave no indication of an oncogenic potential of endosulfan in mice. The NOEL is 6 ppm, equivalent to a mean daily test substance intake of 0.84 mg/kg body weight in male and 0.97 mg/kg body weight in female mice. DISCUSSION
Daily dietary applications of endosulfan technical to Sprague-Dawley rats at 3, 7.5, 15 and 75 ppm for 104wk did not result in overt toxicity. The top concentration met the criteria for the maximum tolerated dose with regard to the point 'significant decrease of body weight gain' (US EPA, 1988) in both sexes. No increase of tumour frequencies compared with control was noticed in either sex. This result is in general agreement with the results of the bioassay of endosulfan performed by the National Cancer Institute (1978). In this study, male and female Osborne-Mendel rats were fed, in two dose groups, diets with increasing and decreasing contents of endosulfan, interrupted by periods of feeding control diet only. The contents ranged from 0 to 600 ppm (low-dose males), 0 to 1200 (high-dose males), 0 to 300 ppm (low-dose females) and 0 to 600 ppm (high-dose females) and finally resulted in time-weighted average high and low dietary concentrations of 952 and 408 ppm for the male rats, and 445 and 223 ppm for the female rats. The application of these doses led to overt toxicity in both sexes and to premature termination of both male dose groups due to high early mortality. Since the survival in males was not sufficient, an evaluation of the carcinogenic potential of endosulfan was not possible for this sex. In female Osborne-Mendel rats, however, the survival was sufficient for the conclusion that endosulfan has no carcinogenic potential in female rats. Despite the fact that this old study was highly overdosed in terms of maximum tolerated dose, there are no findings contradictory to those previous results in the new study presented here. Daily dietary applications of endosulfan technical to N M R I mice at 2, 6 and 18 ppm for 24 months did not result in overt toxicity. The criterion to meet the
Endosulfan: chronic toxicity and carcinogenicity maximum tolerated dose was 'increased mortality' (US EPA, 1988) in the top-concentration females. There was no increase of tumour frequencies compared with controls in either sex. This result is also in agreement with the study performed by the National Cancer Institute (1978). In a study protocol similar to the protocol described above for the Osborne-Mendel rat, male and female B6C3F~ mice were fed time-weighted average high and low dietary concentrations of 6.9 and 3.5 ppm (males) and 3.9 and 2.0 ppm (females), with ranges from 3.0 to 3.5 ppm (low-dose males), 6.0 to 7.0 ppm (high-dose males), 1 5 to 2.0 ppm (low-dose females) and 3.0 to 4.0 (high-dose females). The early mortality observed in the male mice prohibited an evaluation of the carcinogenic potential of endosuifan in this sex; in female B6C3Ft mice, however, endosulfar, proved to be non-carcinogenic. The preliminary study, in which endosulfan was fed for approximately 18 months to both sexes of two hybrid mouse strains (C57BL/6 X C3H/Anf and C57BL/6 X AKR) at 3 ppm resulted in no significant increase of tumour incidences (Innes et al., 1969). As in both rat studies (the old National Cancer Institute study and the new rat study presented here), there were also no conflicting results between the old National Cancer Institute study and the new mouse study presented her,~. Warngard and co-workers from the Karolinska Institute, Stockholm, Sweden, investigated the effects of endosulfan technical, its isomers ~- and fl-endosulfan, and its metabo]ites on gap junctional intercellular communication (GJIC) and their role in tumour promotion in several in vitro experiments (Flodstrom et al., 1988; Fransson-Steen and Warngard, 1992; Kenne et al., 1994; Ruch et al., 1990) and two in vivo short-term carcinogenicity tests (Flodstrom et al., 1988; Fransson-Steen et al., 1992). On the basis of their results, the authors concluded that endosulfan and its isomers are able to inhibit GJIC in vitro and that c~-endosulfan is able to enhance the frequency of -glutamyl transpel:,tidase-altered hepatic foci in vivo. Although the scientific value of these studies is acknowledged, their value for predicting the carcinogenic potential is a subject of debate within the scientific community. The fact that none of the valid lifespan carcinogenicity studies revealed treatment-related differences between control and treated animals in any of the relevant tumour data, together with the absence of any mutagenic potential in all mutagenicity tests (WHO/FAO, 1990), makes it reasonable to conclude that endosulfan has no carcinogenic potential. REFERENCES
Barnard A. V., Atkirson J. S., Heywood R., Street A. E., Gibson W. A., Rao R., Offer J. M., Gopinath G. and Almond R. H. 0984) Endosulfan--active ingredient technical: 13-week toxicity study in mice. HST 229/831052.
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Huntingdon Research Centre Ltd, Huntingdon, UK (unpublished report). Barnard A. V., Jones D. R., Powell L. A. J., Heywood R., Street A. E., Gibson W. A., Gopinath G., Majeed S. K. and Almond R. J. (1985) Endosulfan--active ingredient technical: 13-week toxicity study in rats followed by a 4 week withdrawal period. HST 230/84176. Huntingdon Research Centre Ltd, Huntingdon, UK (unpublished report). Dikshith T. S. S. and Datta K. K. (1978) Endosulfan. Lack of cytogenetic data effects in male rats. Bulletin of Environmental Contamination and Toxicology 20, 826-833. Donaubauer H. H., Leist K.-H. and Kramer M. (1985) Endosulfan, substance technical: 42-day feeding study in mice. Report No. 88.0574. Hoechst Aktiengesellschaft, Pharma Development Corporate Toxicology, Frankfurt, Germany (unpublished report). Edwards J. A. Reid Y. J., Offer J. M., Almond R. H. and Gibson W. A. (1984) Effects of endosulfan on reproductive function of multiple generations in the rat. HST 204/83768. Huntingdon Research Centre Ltd, Huntingdon, UK (unpublished report). Flodstrom S., Warngard L., Hemming H., Fransson R. and Ahlborg U. G. (1988) Tumour promotion related effects by the cyclodiene insecticide endosulfan studied in vitro and in vivo. Pharmacology and Toxicology 62, 230-235. Fransson-Steen R., Flodstrom S. and Warngard L. (1992) The insecticide endosulfan and its two stereoisomers promote the growth of altered hepatic foci in rats. Carcinogenesis 13, 2299-2303. Fransson-Steen R. and Warngard L. (1992) Inhibitory effects of endosulfan on gap junctional intercellular communication in WB-F344 rat liver cells and primary rat hepatocytes. Carcinogenesis 13, 657462. Gupta P. K., Chandra S. V. and Saxena D. K. (1978) Teratogenic and embryonic effects of endosulfan in rats. Acta pharmacologica et toxicologica 42, 150-152. Innes J. R., Ulland B. M., Valerio M. G., Petrucelli L., Fishbein L., Hart E. R., Pallotta A. J., Bates R. R., Falk H. L., Gart J. J., Klein M., Mitchell I. and Peters J. (1969) Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: a preliminary note. Journal of the National Cancer Institute 42, 1101-1114. IVA (1990) Wirkstoffe in Pflanzenschutz -und ScMidlingsbekiimpfungsmitteln: physikalisch-chemische und toxikologische Daten. 2rid Ed. BLV Verlagsgesellschaft, Munich. Kenne K., Fransson-Steen R., Honkasalo S. and Warngard L. (1994) Two inhibitors of gap junctional intercellular communication, TPA and endosulfan: different effects on phosphorylation of connexin 43 in the rat liver epithelial cell line, IAR 20. Carcinogenesis 15, 1161-1165. National Cancer Institute (1978) Bioassay of Endosulfanfor Possible Carcinogenicity. Technical report No. 62. DHEW Publication No. (NIH) 78-1312. US Department of Health, Education and Welfare, Bethesda, MD. Ruch R. J., Fransson R., Flodstrom S., Warngard L. and Klaunig J. E. (1990) Inhibition of hepatocyte gap junctional intercellular communication by endosulfan, chlordane and heptachlor. Carcinogenesis 11, 1097-I 101. US EPA (1988) Selection of a Maximum Tolerated Dose (MTD) in oncogenicity studies. EPA No, 540/09-88-003. Environmental Protection Agency, Research Triangle Park, NC. Usha Rani M. V., Reddy O. S. and Reddy P. P. (1980) Mutagenicity studies involving eldrin, dieldrin, endosulfan, dimethoate, phosphamidon, carbaryl and ceresan. Bulletin of Environmental Contamination and Toxicology 25, 277-282. WHO (1984) Endosulfan. Environmental Health Criteria 40. pp. 25-26. World Health Organization, Geneva. WHO/FAO (1990) Pesticide Residues in Food--1989. FAO
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Plant Production and Protection Paper 100/2. pp. 95-115. Food and Agriculture Organization of the United Nations, Rome. Yang R. S. H., Huff J., Germolec D. R., Luster M. I., Simmons J. E. and Secly J. C. 0989) Biological issues in
extrapolation. In Carcinogenicity and Pesticides. Principles, Issues, and Relationships. ACS Symposium Series 414. Edited by N. N. Ragsdate and R. Menzer. pp. 142-163. American Chemical Society, Washington, DC.
Pergamon
0278-6915(95)00063-1
Fd Chem. Toxic. Vol. 33, No. 11, pp. 941-950, 1995 Copyright © 1995 ElsevierScienceLtd Printed in Great Britain. All fights reserved 0278-6915/95 $9.50 + 0.00
Chronic Toxicity and Carcinogenicity Studies with the Insecticide Endosulfan in Rats and Mice R. H A C K * t , E. E B E R T a n d K . - H . L E I S T Hoechst Schering AgrEvo GmbH, Toxicology and *Hoechst Aktiengesellschaft, Pharma Development Ccrporate Toxicology, D-65795 Hattersheim, Mainzer Landstrasse 500, Germany
(Accepted 16 May 1995) Abstract--The insecticide endosulfan was evaluated for chronic toxicity and carcinogenicity in long-term feeding studies in both Sprague-Dawley rats and NMRI mice. Dietary concentrations of the test substance were adminis~:ered to rats at 0, 3, 7.5, 15 and 75 ppm and to mice at 0, 2, 6 and 18 ppm for 24 months each. In the rat study, only the treatment with the highest dose caused a significant reduction of body weight gains of the males and females at 75 ppm. The increased incidence of enlarged kidneys seen at autopsy at 75 ppm in females, and the slightly increased incidence of progressive glomerulonephrosis and slightly increased incidence of renal aneurysms seen histopathologically in the 75 ppm males, make the kidney the target organ in rats. On the basis of these findings a dietary concentration of 15 ppm is considered to be the no-observed-effect level (NOEL) in rats, equivalent to a daily test substance intake of 0.6 mg/kg body weight in males and 0.7 mg/kg body weight in females. In the mouse study, the treatment with 18 ppm caused a significant increase of mortality in the females and a slight (in the first third of the study, significant) reduction of body weight gain in males. Since there were no other substance-related findings, the dietary concentration of 6 ppm is considered to be the NOEL in mice, equivalent to a daily test substance intake of 0.84 mg/kg body weight in males and 0.97 mg/kg body weight in females. An evaluation of all relevant tumour data gained in both studies revealed no differences between control and treated groups. It was concluded, therefore, that endosulfan has no carcinogenic potential.
1NTRODUCTION
strains, in a yeast forward mutation test using Schizosaccharomyces pombe, in a yeast gene convers i o n / D N A repair test using Saccharomyces cerevisiae strain D4, in a mouse lymphoma test using mouse L51786 T K ± cells, in a D N A repair test using Bacillus subtilis strain H17 and M45 (rec-), in unscheduled D N A synthesis tests using primary rat hepatocytes and the human cell line A549 and in a chromosome aberration test using cultured human lymphocytes ( W H O / F A O , 1990). There was also no indication of a mutagenic potential of endosulfan in in vivo mutagenicity tests--micronucleus tests using bone marrow of N M R I ( W H O / F A O , 1990) and Swiss Albino mice (Usha Rani et al., 1980)--and an in vivo cytogenicity test using bone marrow and spermatogonia of male albino rats (Dikshith and Datta, 1978). Endosulfan has been already investigated for possible carcinogenicity in mice and rats as part of the Carcinogenesis Testing Program of the National Cancer Institute (Innes et al., 1969; National Cancer Institute, 1978). The preliminary mouse study resuited in no significant increase in tumours (Innes et al., 1969); in the subsequent studies in mice and rats, mortality in both male mice and rats was ultimately extremely high (National Cancer Institute, 1978). For this reason, these studies were classified by
Endosulfan is an insecticide used worldwide in agriculture, viticulture and horticulture. It was first registered by Hoechst in 1958 under the trade mark Thiodan in Germany. The first registration in the United States was in 1960. After oral application to rats, endosulfan has an LDs0 from 18 to 355mg/kg body weight (WHO, 1984) or from 40 to l l 0 m g / k g body weight (IVA, 1990), depending on the sex of the animal and the vehicle used. Signs of acute intoxication include neurological manifestations, such as sedation, hyperactivity, muscular twitching, ataxia and convulsions (IVA, 1990; W H O , 1984). Endosulfan is irritating to the eyes and skin, and not sensitizing in the guinea pig (IVA, 1990). Endosulfan is not teratogenic to rats and rabbits (Gupta et al., 1978; W H O / F A O , 1990) and has no effect on rat reproduction ( W H O / F A O , 1990). The compound was not mutagenic in vitro in an Ames test using various Salmonella typhimurium tAuthor for correspondence. Abbreviations: GJIC = gap junctional intercellular communication; GOT = glutamic--oxaloacetic transaminase; GPT= glutamic-pyruvic transaminase; NOEL = noobserved-effect level. 941
942
R. Hack et al.
the NTP (Yang et al., 1989) and W H O / F A O (1990) as inadequate for assessing the carcinogenic potential of endosulfan. The purpose of this paper is to present the data of valid up-to-date studies that were performed to find a sound basis for evaluating the chronic toxicity and the carcinogenic potential of endosulfan. MATERIALS AND METHODS
Test substance
Endosulfan [6,7,8,9,10,10-hexachloro-l,4,5a,6,9,9ahexahydro - 6,9- methano- 2,4,3 - benzo- dioxathiepin3-oxide (IUPAC; CAS Reg. No. 115-29-7)] was supplied by the Agricultural Division, Hoechst Aktiengesellschaft and identified by analysis certificates for the content of active ingredient and impurities (see Table 1). Both studies were performed with the same batch. The stability was confirmed by regular re-analysis. None of the analyses revealed a content of less than 96.7% (w/w). Rat study
The study was performed at Huntingdon Research Centre Ltd (Huntingdon, UK). Sprague-Dawley rats [stock: Crl:Cd (SD) BR], were obtained from Charles River (Portage, MI, USA). They were approximately 6 wk old at the start of treatment and had a mean body weight, after a 1-wk adaptation period, of 129 g (males) and 109 g (females). The animals were housed five to a cage on a wire mesh floor. The doses used throughout this study were based on the results of a 13-wk feeding study (Barnard et al., 1985; WHO/FAO, 1990) and on a multigeneration study (Edwards et al., 1984; WHO/FAO, 1990).
In the 13-wk feeding study, Sprague-Dawley rats [stock: CrI:CD (SD) BR] received a diet containing 0, 10, 30, 60 and 360ppm endosulfan, equivalent to daily doses of 0.64, 1.92, 3.85 and 23.41 mg/kg body weight in males and 0.75, 2.26, 4.59 and 27.17 mg/kg body weight in females. From 60ppm onwards, slightly reduced body weight gains, haematological changes and increases of relative kidney weights (males, females at 360 ppm) were observed. In the multigeneration study, feeding of Sprague-Dawley rats [stock: Crl:CD (SD) BR] with daily concentrations of 0, 3, 15 and 75 ppm endosulfan, equivalent to approximately 0.2, 1.0 and 6.0 mg/kg body weight, resulted in no clinical findings in any group and in slight but not statistically significant impairment of body weight gains in the 75 ppm group. The maximum tolerated dose, based on the reduction of body weight gain (US EPA, 1988), was considered to be around 75 ppm when administered for 104 wk. The study design consisted of five groups (including one untreated control group) that were fed endosulfan technical grade with the daily diet (Labsure Animal Diet No. 2, SDS Diets Ltd, Witham, UK) in concentrations of 0, 3, 7.5, 15 and 75 ppm for 104 wk. One premix was prepared each week by dissolving the test material in acetone (final concentration in the premix, 0.4%), mixing the acetone with corn oil (final concentration in the premix, 2%) and mixing the acetone-corn oil with basal diet. The acetone was removed from the premix using a rotary evaporator at a temperature of 40°C, the mixture being left in the rotary evaporator flask until a constant weight was achieved. All the treated diet was made from the premix containing the test material, corn oil and acetone (the acetone having theoretically been
Table 1. Characterizationof the test substance used throughout both studies Common name: Chemical name (IUPAC) CAS Reg. No. Content [% (w/w)]* Analysis of the active ingredient:
Endosulfan: 6,7,8,9,10-Hexachloro-1,4,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepin-3-oxide
115-29-7
96.7-97.9
Endosulfan-ether: 4,5,6,7,8,8-Hexachloro-1,3,3a,4,7,7a-hexahydro-4,7-methano-isobenzofurane
3369-52-6
0.2-0.3
Endosulfan-diol: 1,4,5,6,7,7-Hexachloro-bicyclo[2.2.1.]bept-5-ene-2,3-dimenthanol
2157-19-9
0.2-0.3
Analysis of impurities:
Endosulfan-lactone: 3868-61-9 4,5,6,7,8,8-Hexachloro-1,3,3a,4,7,7a-hexahydro-4,7-methano-isobenzofurane-l-one Endosulfan-acetate: 1,4,5,6,7,7-Hexachloro-bicyclo[2.2.1.]bept-5-ene-2,3-dimethyl-diacetate
18992-36-4
0.4 < 0.2
Octachlorocyclopentene
--
0.1- < 0.1
Solvent residues(e.g. toluene)
--
0.2-<0.1
NaCI (measuredas chloride)
--
<0.05
Epoxidizedsoybeanoil
--
0.33-0.34
*Range of several analyses.
Endosulfan: chronic toxicity and carcinogenicity i
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During the first 4 wk, all animals were observed daily for clinical signs of toxicity, and subsequently once weekly. Examination for palpable masses was carried out every week. Mortality was recorded twice daily. Body weights were determined pre-test, at the beginning of treatment and once a week thereafter. Food consumption was measured weekly and water consumption during wk 11, 26 and 50 (chronic toxicity groups only). Ophthalmoscopic examination was carried out pre-test and during wk 50 and 104 in all animals of the control and high-concentration groups. Clinical laboratory investigations were performed in the chronic toxicity group only; for the last examinations, the sample size of 10 animals per sex was reached, where necessary, by replenishment with animals from the tumorigenicity groups. Blood samples were collected (chronic toxicity groups only) from the orbital sinus during wk 13, 26, 52, 78 and 103 after food removal overnight, and analysed for haematocrit, haemoglobin, red blood cell count, platelet count, total and differential white blood cell count, thrombin time, total protein, albumin, globulin, urea nitrogen, creatinine, sodium, potassium, calcium, inorganic phosphorus, chloride, glucose, alkaline phosphatase, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), cholesterol and total bilirubin. Urine was collected during wk 12, 25, 51, 77 and 102 after water removal
Fig. 1. Percentage survival of (a) male and (b) female rats fed endosulfan [0 ( - - ) , 3(. . . . ), 7.5 (. . . . . ), 15 (-.-) or 75 ppm (. . . . )] in the diet for 24 months.
i
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100
90 - (a)
completely removed) by direct dilution of the premix with further amounts of standard diet for a minimum of 7 min in a double cone blender. The amount of premix used increased from the lowest-concentration diet to the high-concentration diet. The amount of corn oil (and the hypothetical amount of acetone remaining if none had been removed) in the highest concentration diet was calculated. For the control diet, a premix containing basal diet corn oil at 2% and acetone at 0.4% was prepared such that the concentration of corn oil and the hypothetical concentration of acetone in the final control and high-concentration diets never exceeded a value of 0.5% for corn oil ',and 0.1% for acetone. The final diets were stored at room temperature in the animal rooms. The stability, homogeneity and accuracy of mixing were determined prior to the start of treatment and checked in the first week and after 3monthly intervals. A constant concentration was administered to the treated groups throughout the study. Each group consisted of 50 animals per sex, intended primarily fc,r tumorigenicity evaluation. For the purpose of evaluating chronic toxicity, a further 20 rats per sex per dose were scheduled for blood sampling at scheduled intervals and for killing after 104 wk of treatment and were therefore not included in the tumorigenicity evaluation.
943
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60
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65
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91
104
Study
wk
Fig. 2. Percentage survival of (a) male and (b) female mice fed endosulfan [0 ( - - - ) , 2(. . . . ), 6 (. . . . ) or 18 ppm (-. -)] in the diet for 104wk.
944
R. Hack et al. Table 2. Statistical analysis of body weight changes of rats fed endosulfan for a period of 24 months Body weight changes (g) Male dose groups (ppm)
Treatment period (wk)
0
3
7.5
Female dose groups (ppm) 15
75
0-6
230 (30.0) --
233 (33.3) [101]
234 (36.7) l 102]
226 (30.6) [98]
15
229 (30.7) [I 00]
75
113 (20.7) --
0
110 (18.3) [96]
3
113 (17.6) [100]
7.5
108 (16.6) [96]
104"* (15.5) [92]
6-18
160 (28.0) --
157 (31.8) [98]
159 (38.7) [99]
145"* (32.2) [91]
144"* (31.6) [901
69 (17.2) --
66 (19.0) [96]
69 (19.2) [100l
68 (18.2) 199]
65 (15.0) I94]
18~4
200 (55.9) --
203 (72.8) [102]
194 (76.9) [97]
193 (74.5) [97]
163"* (76.5) [82]
153 (67.4) --
146 (57.7) [95]
153 (59.6) [100]
143 (57.3) [93]
125"* (61.7) [82]
0--64
590 (91.5) --
594 (109.3) [101]
586 (127.6) [99]
564 (107.8) [96]
536** (105.8) [91]
337 (88.1) --
322 (79.3) 196]
334 (82.6) [99]
318 (77.4) [94]
294** (76.3) [87]
64-104
- 11 (103.4)
13 (99.7)
-9 (113.0)
9 (102.4)
-43 (107.3)
94 (117.1)
69 (101.9)
80 (67.4)
82 (76.9)
56 (64.6)
0-104
580 (124.0) --
570 (125.4) [98]
531 (131.3) [92]
525 (115.4) [911
479** (91.4) 183]
398 (104.7) --
350 (107.3) [88]
414 (84.7) [104]
363 (91.9) [91]
328* (100.0) [82]
Values are means (g); +standard deviation in parentheses; percentage of control in square brackets. Asterisks indicate significant differences from control (*P < 0.05; **P < 0.01; Williams' test).
overnight, and analysed for volume, pH, specific gravity, protein, total reducing substances, glucose, ketones, bile pigments, urobilinogen, haem pigments and sediment. Complete gross autopsies were performed on all animals. Selected organs were weighed. Tissues-including macroscopically abnormal tissues from all animals of the control and high-dose group, tissues of all animals that had died or were killed in e x t r e m i s during the course of the study, any macroscopically abnormal tissue in any group, and lungs, liver and kidneys of all groups at terminal killing--were fixed in 10% formaldehyde solution and embedded in paraffin wax. Section cut at 4/zm were stained with haematoxylin and eosin; in addition, frozen sections of liver fixed in 10% formaldehyde solution and cut at 12 #m were stained for fat with Oil Red O.
4 wk and a mean body weight after a 1-wk adaptation period of 23 g (males) and 22.5g (females). The animals were kept individually in Macrolon cages (type I) with wire mesh tops, with standardized and sterilized granulated softwood bedding. The doses used throughout this study were based on the results of a 3-month subchronic oral toxicity study (Barnard et al., 1984; WHO/FAO, 1990) and of a supplementary 6-wk feeding study (Donaubauer et al., 1985; WHO/FAO, 1990). In the subchronic study CD-I mice [stock: CrI:CD-I(ICR)BR] were fed diets containing the test material at doses of 2, 6, 18 or 54ppm, equivalent to 0.24, 0.74, 2.13 and 7.3 mg/kg body weight in males and 0.27, 0.80, 2.39 and 7.5 mg/kg body weight in females. Mice receiving 54 ppm exhibited convulsions and salivation, and 12 of 20 males and 10 of 20 females died within 6 wk of study initiation. No mortality or other toxic effects were observed at lower levels. The 6-wk study with NMRI mice [stock: Hoe:NMRKf (SPF71)] given 18 ppm with the diet, equivalent to 3.7 and 4.6 mg/kg body weight in males and females, respectively, was performed to check the sensitivity of the NMRI mice to endosulfan. The duration was limited to 6 wk since
M o u s e study
The study was performed at Hoechst Aktiengesellschaft, Pharma Development Corporate Toxicology, Frankfurt, Germany. NMRI mice [stock: Hoe:NMRKf (SPF71)] from the Hoechst breeding colony were used. They had an age of about
Table 3. Relative incidence of selected non-neoplastic findings in male rats after feeding of endosulfan for 104 wk compared with historical control data
Dose groups~: (ppm) Findings
0
Progressive 0.286 glomerulonephrosis Aneurysm 0.143
3
7.5
0.257
0.314
0.342 0.428t
15
75
Historical rang~ 0.100-0.380
0.086
0.243
0.143 0.271"
0.040-0.180
*n = 70; tumorigenic + chronic toxicity group. §Range of six studies with n = 50 animals each. Asterisk and dagger indicate significant differences from controls (*P <0.05; tP < 0.06; Fisher's exact test).
E n d o s u l f a n : c h r o n i c toxicity a n d c a r c i n o g e n i c i t y
945
Table 4. Mcidence of neoplastic lesions in rats after administration of endosulfan for 104 wk (n = 50) Male dose groups (ppm)
Female dose groups (ppm)
Organ/tumour type*
0
3
7.5
15
75
0
3
7.5
15
75
Multicentric tumours Lymphosarcoraa Myeloid leukaemia Histiocytic sarcoma
0 0 0
I 0 0
0 1 0
0 0 0
0 0 0
0 0 1
0 0 0
0 0 1
2 0 0
1 0 0
Liver Haemangioma Haemangiosarcoma Malignant liver cell tumour Cholangioma
0 0 1 1
0 0 1 0
1 0 1 0
0 0 1 0
0 0 2 0
0 0 0 0
1 0 0 0
1 1 0 0
0 1 0 0
0 0 0 0
Kidney Renal liposarcoma Renal carcinoma Renal lipoma Mesenchymal tumour
1 0 0 0
0 1 0 0
1 0 0 0
0 0 1 0
l 1 0 0
1 0 0 0
0 0 0 0
0 1 0 0
0 0 0 1
0 0 0 0
Lungs Pulmonary adenocarcinoma Pulmonary adenoma
0 0
1 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 1
Brain Granulocytic meningioma Astrocytoma
0 0
1 1
0 0
0 l
0 0
0 1
0 0
0 0
0 0
0 I
Spleen Haemangioma Haemangiosarcoma Fibrosarcoma
0 0 0
0 l 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 1
0 0 0
0 0 0
1 0 0
Pancreas Islet cell carcinoma Islet cell adenoma Islet cell aderoma (two)
3 8 0
8 6 0
3 9 0
5 11 3
5 4 0
0 0 0
0 5 0
0 0 0
0 5 0
0 5 0
Thyroids Parafonicular cell carcinoma Follicular carcinoma Follicular adenoma
3 0 0
0 3 0
0 0 4
0 0 0
3 0 5
0 0 0
3 0 0
0 0 0
0 0 0
0 0 0
Adrenals Phaeochromccytoma
3
0
0
3
0
0
0
0
0
0
0 23
0 18
0 16
0 21
0 27
3 31
0 31
0 39
0 34
0 32
Skin Fibroma Dermal fibro:na Keratoacantl" oma
0 5 0
0 3 4
0 0 5
3 0 4
0 0 3
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
Prostate Prostatic adeaocarcinoma
1
1
0
0
0
Testes Interstitial cell tumour
0
2
2
2
1
Epididymides Mesothelioma
0
0
0
1
0
Uterus Fibrosarcoma Endometrial sarcoma Squamous cell carcinoma Uterine adenocarcinoma
-----
-----
1 1 0 0
1 1 0 0
0 1 0 0
0 0 0 0
0 0 I 1
Ovaries Tubular adenoma
--
--
0
2
0
1
0
Cervix Stromal sarcoma
--
--
0
0
0
0
0
Subcutaneous mass Lipoma Fibroma Mammary fibroma Mammary fibroadenoma Mammary fibroadenoma with atypia Mammary adenoma Mammary adenocarcinoma
5 3 0 2 0 0 0
7 0 0 2 0 0 0
10 3 0 1 0 0 1
3 3 0 1 0 0 1
3 3 0 0 0 0 0
0 3 0 34 8 0 14
0 4 4 34 3 3 12
0 3 0 36 4 0 14
0 4 0 29 0 3 9
0 0 5 31 3 0 10
Mammary gland Basal cell carcinoma
0
1
0
0
0
0
0
0
0
0
Pituitary Carcinoma in pars anterior Adenoma in pars anterior
m
m
m
_
E
m
_
E
m
m
*Only tumours of the main organs, possible target organs and those for which the incidence was greater than one in any of the groups are presented. Statistical significance was tested using Mante!-Haenszel's test and Fisher's exact test.
946
R. Hack et al. Table 5. Cumulative mortality and percentage survivalt in mice (n = 60) fed endosulfan for 24 months Male dose groups (ppm) Female dose groups (ppm) Study wk 0 2 6 18 0 2 6 18 1-26 3 (95) I (98) I (98) 2 (97) 3 (95) 2 (97) 0 (100) 1 (98) 27-52 5 (92) 8 (87) 10 (83) 15 (75) 4 (93) 2 (97) 8 (87) 12 (80) 53-78 1 2 ( 8 0 ) 1 4 ( 7 7 ) 1 6 ( 7 3 ) 17(72) 11 (82) 1 0 ( 8 3 ) 1 6 ( 7 3 ) 23(62) 79-end 27 (55) 33 (45) 37 (38) 35 (42) 33 (45) 36 (40) 38 (37) 43 (28)* tPercentage survival in parentheses. Asterisk indicates significantdifferencefrom control (*P < 0.05; Kaplan Meier estimate and log rank test).
the only s y m p t o m s (i.e. clinical signs of toxicity and mortality) had appeared during this period of treatm e n t in the 3 - m o n t h study. Two of 10 females fed diet containing 18 p p m died on days 28 and 38; no other effects were reported. The m a x i m u m tolerated dose based on the mortality (US EPA, 1988) was considered to be a r o u n d 18 p p m when fed for a period of 24 months. The study design consisted o f four groups (including one untreated control group) t h a t were fed endosulfan technical grade with the daily diet (maintenance diet 1321, A l t r o m i n International, Lage, G e r m a n y ) in c o n c e n t r a t i o n s of 0, 2, 6 and 18 p p m for 24 months. A premix for each dose group was prepared at 14-day intervals by dissolving the test substance in sesame oil (final concentration in the premix, 2%), then by mixing with the basal diet so that the premix c o n t a i n e d the 14-fold c o n c e n t r a t i o n of test substance of the final concentration. The final dietary mixtures were prepared fresh once weekly by direct dilution of the premixes with 13-fold a m o u n t s of the basal diet for 30 min in a p l o u g h s h a r e mixer. C o n t r o l animals received n o r m a l diet prepared from a premix with 2 % sesame oil without addition o f test substance by admixture of 13-fold the same a m o u n t o f basal diet. The final diets c o m p o s e d of test substance (not in the control), of a fixed portion of 0.14 corn oil a n d basal diet were stored at r o o m t e m p e r a t u r e in the animal rooms. The stability, concentrations a n d h o m o g e n e i t y o f mixtures were determined prior to the start of t r e a t m e n t and checked four times during the course of the study. The four groups each consisted o f 60 animals per sex a n d dose and were scheduled for terminal killing after 24 months. A further 10 animals per sex a n d
dose were scheduled for interim killing after 12 a n d 18 months, respectively for each group, and were therefore not included in the tumorigenicity evaluation. Behaviour, general health condition a n d mortality were checked twice daily (at weekends a n d on public holidays once daily). Once a week the animals were examined for neurological disturbances, opacity of the refracting media o f the eyes, i m p a i r m e n t of dental growth a n d changes in the oral mucosa. Body weights and food c o n s u m p t i o n were determined once weekly. E x a m i n a t i o n for palpable masses was performed monthly, a n d subsequently twice m o n t h l y from m o n t h 6 onwards. Blood samples were taken from 10 non-starved animals per sex a n d dose after 6 m o n t h s (intermediate, using the 12-month interim killing group), 12 m o n t h s (at interim killing), 18 m o n t h s (at interim killing) and 24 m o n t h s (at terminal killing). The samples were analysed for red blood cell count, haemoglobin, haematocrit, total a n d differential white blood cell count, platelet c o u n t a n d reticulocyte count. The serum enzymes alkaline phosphatase, G O T and G P T were m e a s u r e d after killing only. Complete gross autopsies were performed on all animals. Selected organs were weighed. T i s s u e s - including macroscopically a b n o r m a l tissues from all animals of any group, tissues of all animals that had died or were killed in extremis during the course of the study, any macroscopically a b n o r m a l tissue in any group, and lungs, liver and kidneys of all groups at terminal killing--were fixed in 10% formaldehyde solution a n d e m b e d d e d in paraffin wax. Sections cut at 4/~m were stained with haematoxylin a n d eosin.
Table 6. Representative results of mean body weightt of mice (n = 60) fed endosulfan for 24 months Male dose groups (ppm) Female dose groups (ppm) Study wk 0 2 6 18 0 2 6 18 1 27(_+2) 26(_+2) 27(_+2) 25(+_2) 23(-+1) 23(-+1) 23(_+1) 23(-+1) 13 35(_+3) 35(-+3) 35(-+3) 34(_+3)* 28(-+2) 28(_+2) 29(_+2) 29(_+2) 26 37(_+3) 37(_+3) 37(_+4) 35(_+4)* 30(_+2) 30(_+2) 30(_+2) 31 (_+2) 52 38(_+4) 38(_+4) 39(_+4) 37(-+3) 32(_+2) 32(_+2) 32(_+3) 32(_+4) 78 39(_+3) 38(_+3) 38(_+3) 37(_+4) 34(_+4) 33(_+3) 34(_+3) 34(-+2) 92 38(-+3) 38(_+3) 37(_+3) 36(_+4) 34(+3) 33(_+3) 34(_+3) 34(_+4) 104 37(_+3) 37(_+3) 36(_+3) 35(_+4) 34(_+3) 33(_+3) 34(_+4) 34(_+4) t -+ Standard deviation in parentheses. Asterisks indicate significantdifferencesfrom controls (*P < 0.05; statistical analysis was carried out with a program package including procedures such as parametric method by Dunnett or Sidak and distribution-frce method by Nemenyi/Dunnett or Nemenyi/Sidak).
947
Endosulfan: chronic toxicity and carcinogonieity Table 7. Incidence of selected neoplastic lesions in mice after administration of endosulfan for 104 wk (n = 60) Male dose groups (ppm)
Female dose groups (ppm)
0
2
6
18
0
2
6
18
11 I 3 1 0
13 1 2 0 0
18 1 1 0 0
16
22
25
25
15
1
2
3
1
1
0 0 0
2 0 0
1 0 0
0 0 I
2 0 0
0 0 0 1 1
0 0 1 0 0
1 0 0 0 0
I 2 0 1 0
0 0 0 0 0
0 0 0 0 0
0 0 1 0 0
0 0 0 1 0
Lungs Pulmonary adenoma Two pulmonary adenomas Pulmonary adenocarcinoma
10 1 2
5 0 2
5 0 2
5 0 3
2 0
3 1
2 0
2 0
1
4
2
0
Adrenals Corlical adenoma
13
12
6
10
0
0
0
0
Pituatary Adeaoma in pars anterior
0
0
0
0
6
7
1
0
Testes Inte::stitial cell tumour
2
4
1
0
Organ/l:umour type* Multice:ntdc tumours Lymphosarcoma Pleomorphic lymphosarcoma Histiacytic sarcoma Lymphoid leukaemia Myeloid leukaemia Liver Benign liver cell tumour Haemangioma Haemangiomas Haemangiosarcoma Anaplastic sarcoma
Ovaries Tubular adenoma Papillary cystadenoma Granulosa cell tumour Thecoma Lut(;al cell tumour Haemangioma Haemangiosarcoma Sex cord stromal tumour
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
5 0 8 3 9 I 0 I
3 3 I0 2 5 2 I I
2 3 II 2 2 0 0 0
4 2 13 I I 0 0 0
Uterus An~.plastic carcinoma Leiomyosarcoma End ometrial sarcoma Fibrosarcoma naemangioma Haemangiosarcoma
. . . . . .
. . . . . .
. . . . . .
. . . . . .
1
0
0
0
1
0
0
1
1
0
0
0
0 0 0
0 I 1
0 0 0
I 1 0
Subcutaneous mass Mammary adenocareinoma Mammary fibroadenoma with atypia Mammary adenoacanthoma Fibrosarcoma Haemangiosarcoma
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 I 0
1
0
3
0
0
0
1
0
1
0
0
0
0 0
0 0
0 0
0 I
*Only tumours of the main organs, possible target organs and those for which the incidence was greater that one in any of the groups are presented. Statistical significance was tested using MantelHaenszel's test and Fisher's exact test.
RESULTS
Rat study T h e m o r t a l i t y rate w a s unaffected by the t r e a t m e n t (Fig.
l). N o
treatment-related
effects o n clinical
condition, food consumption, water consumption, h a e m a t o l o g y , clinical c h e m i s t r y , urinalysis a n d o p h t h a l m o s c o p y were a p p a r e n t . T h e overall b o d y w e i g h t gain f o r rats o f either sex receiving 7 5 p p m ,
however, was lower than
that
o f c o n t r o l s d u r i n g the m a i n g r o w t h p h a s e o f the s t u d y ( w k 0--64), :an effect t h a t w a s m o r e s t r o n g l y m a r k e d in the females t h a n in the males. T h e effect FCT33/II--D
o n the g r o w t h o f m a l e s at 75 p p m w a s n o t a p p a r e n t until after w k 6, w h e r e a s the effect o n females at 75 p p m w a s n o t e d f r o m the s t a r t o f t r e a t m e n t . T h e o t h e r d o s e g r o u p s were c o n s i d e r e d n o t to be affected ( T a b l e 2). T h e r e were n o statistically significant effects o n o r g a n w e i g h t s in a n y d o s e g r o u p . T h e m a c r o s c o p i c p a t h o l o g y revealed t r e a t m e n t - r e l a t e d a l t e r a t i o n s in the 75 p p m g r o u p in the f o r m o f a g r e a t e r n u m b e r o f e n l a r g e d k i d n e y s in female rats, a g r e a t e r n u m b e r o f a n e u r y s m s in m a l e r a t s m a i n l y f r o m the c h r o n i c toxicity g r o u p , a n d a g r e a t e r n u m b e r o f e n l a r g e d l u m b a r l y m p h n o d e s w i t h n o clear-cut
948
R. Hack et al.
treatment-related histopathological changes in males mainly from the chronic toxicity group. The histopathological examination revealed, in the highest male group, a minimally increased incidence of progressive glomerulonephrosis and an increased incidence of aneurysms in the kidneys compared with the control and the historical range (Table 3). The findings of macroscopic and histopathological examination make the kidney the target organ, especially in the male rat. There was no indication of any treatment-related effect on the reproductive organs. The neoplastic changes are summarized in Table 4. Among the most commonly occurring tumours were pituitary tumours in male and female rats and mammary tumours in females. The type and frequency of tumours were similar in dosed and control rats and gave no indication of a treatment-related effect. The results of this study gave no indication of any oncogenic potential of endosulfan in rats. The NOEL is 15 ppm, equivalent to a mean daily test substance intake of 0.6 mg/kg body weight in the males and 0.7 mg/kg body weight in the females. Mouse study
There were no treatment-related effects on clinical signs, food and water consumption, clinical chemistry and haematology throughout the study. No differences in the number of palpable masses between dosed and control animals were noted. The mortality rate among the females in the 18 ppm dose group, however, was significantly increased. The cumulative mortality and percentage survival data are shown in Fig. 2 and summarized in Table 5. At study termination after 106 wk the survival rate among the high-dose females was lower than that among the controls (28% v. 45%). Survival among other dosed females and all dosed males was comparable to controls, ranging from 37 to 55%. Survival among dosed animals of the satellite groups killed at 12 months was comparable with that of the controls; no deaths occurred among dosed or control animals scheduled for interim killing at 18 months (data not shown). The development of body weights is shown in Table 6. Body weights of males receiving 18 ppm were slightly but significantly decreased in the first third of the study when compared with concurrent controls; body weights of these animals remained slightly but not significantly decreased thereafter, to study termination. The body weights of other dosed males and all dosed females were similar to those of controls throughout the study. Macroscopic pathology gave no indications of intergroup differences. The organ weights after 24 months did not reveal any statistically significant differences between control and treated animals. At 12-month interim killing the relative lung and ovary weights in the top-dosed females, and after 18-month interim killing the relative liver weights of the topdosed males and ovary weights of the top-dosed
females, were lowered. All these values, however, were within the normal range of this stock of mice and were therefore considered as incidental (data not shown). The histopathological examination revealed no treatment-related changes at any killing point or for any sex and dose. The neoplastic findings are listed in Table 7. The most commonly observed neoplasms were lymphosarcomas and pulmonary adenomas in both sexes, adrenal cortical adenomas in male and granulosa cell tumours and luteal cell tumours in females. The incidence of findings was similar in dosed and control animals. There was no indication of any effect on reproductive organs. All findings were considered to be of a normal age- and stockrelated nature. The results of this study gave no indication of an oncogenic potential of endosulfan in mice. The NOEL is 6 ppm, equivalent to a mean daily test substance intake of 0.84 mg/kg body weight in male and 0.97 mg/kg body weight in female mice. DISCUSSION
Daily dietary applications of endosulfan technical to Sprague-Dawley rats at 3, 7.5, 15 and 75 ppm for 104wk did not result in overt toxicity. The top concentration met the criteria for the maximum tolerated dose with regard to the point 'significant decrease of body weight gain' (US EPA, 1988) in both sexes. No increase of tumour frequencies compared with control was noticed in either sex. This result is in general agreement with the results of the bioassay of endosulfan performed by the National Cancer Institute (1978). In this study, male and female Osborne-Mendel rats were fed, in two dose groups, diets with increasing and decreasing contents of endosulfan, interrupted by periods of feeding control diet only. The contents ranged from 0 to 600 ppm (low-dose males), 0 to 1200 (high-dose males), 0 to 300 ppm (low-dose females) and 0 to 600 ppm (high-dose females) and finally resulted in time-weighted average high and low dietary concentrations of 952 and 408 ppm for the male rats, and 445 and 223 ppm for the female rats. The application of these doses led to overt toxicity in both sexes and to premature termination of both male dose groups due to high early mortality. Since the survival in males was not sufficient, an evaluation of the carcinogenic potential of endosulfan was not possible for this sex. In female Osborne-Mendel rats, however, the survival was sufficient for the conclusion that endosulfan has no carcinogenic potential in female rats. Despite the fact that this old study was highly overdosed in terms of maximum tolerated dose, there are no findings contradictory to those previous results in the new study presented here. Daily dietary applications of endosulfan technical to N M R I mice at 2, 6 and 18 ppm for 24 months did not result in overt toxicity. The criterion to meet the
Endosulfan: chronic toxicity and carcinogenicity maximum tolerated dose was 'increased mortality' (US EPA, 1988) in the top-concentration females. There was no increase of tumour frequencies compared with controls in either sex. This result is also in agreement with the study performed by the National Cancer Institute (1978). In a study protocol similar to the protocol described above for the Osborne-Mendel rat, male and female B6C3F~ mice were fed time-weighted average high and low dietary concentrations of 6.9 and 3.5 ppm (males) and 3.9 and 2.0 ppm (females), with ranges from 3.0 to 3.5 ppm (low-dose males), 6.0 to 7.0 ppm (high-dose males), 1 5 to 2.0 ppm (low-dose females) and 3.0 to 4.0 (high-dose females). The early mortality observed in the male mice prohibited an evaluation of the carcinogenic potential of endosuifan in this sex; in female B6C3Ft mice, however, endosulfar, proved to be non-carcinogenic. The preliminary study, in which endosulfan was fed for approximately 18 months to both sexes of two hybrid mouse strains (C57BL/6 X C3H/Anf and C57BL/6 X AKR) at 3 ppm resulted in no significant increase of tumour incidences (Innes et al., 1969). As in both rat studies (the old National Cancer Institute study and the new rat study presented here), there were also no conflicting results between the old National Cancer Institute study and the new mouse study presented her,~. Warngard and co-workers from the Karolinska Institute, Stockholm, Sweden, investigated the effects of endosulfan technical, its isomers ~- and fl-endosulfan, and its metabo]ites on gap junctional intercellular communication (GJIC) and their role in tumour promotion in several in vitro experiments (Flodstrom et al., 1988; Fransson-Steen and Warngard, 1992; Kenne et al., 1994; Ruch et al., 1990) and two in vivo short-term carcinogenicity tests (Flodstrom et al., 1988; Fransson-Steen et al., 1992). On the basis of their results, the authors concluded that endosulfan and its isomers are able to inhibit GJIC in vitro and that c~-endosulfan is able to enhance the frequency of -glutamyl transpel:,tidase-altered hepatic foci in vivo. Although the scientific value of these studies is acknowledged, their value for predicting the carcinogenic potential is a subject of debate within the scientific community. The fact that none of the valid lifespan carcinogenicity studies revealed treatment-related differences between control and treated animals in any of the relevant tumour data, together with the absence of any mutagenic potential in all mutagenicity tests (WHO/FAO, 1990), makes it reasonable to conclude that endosulfan has no carcinogenic potential. REFERENCES
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